Agro-waste as a substrate for the production of pullulanase by Penicillium viridicatum under solid-state fermentation

One of the key enzymes utilized in the food industry is pullulanase. But its major drawbacks are its low yield and high production costs. In this regard, the current research aims to screen agro-waste substrates for optimal pullulanase production in solid-state fermentation. Of various agro-wastes used as a substrate, the maximum enzymic activity (9.74 U/gds) was observed in a medium based on 5 g of green gram husk and incubated for 3 days at 30 °C. The effects of 16 different nutrients on the yield of pullulanase production were studied using the Plackett–Burman experimental design. The incorporation of FeSO4, MnSO4, and MgSO4 into the pullulanase production medium significantly increased the yield and showed a 5.7-fold increase (56.25 U/gds) in comparison with the unoptimized media. The Box-Behnken experimental design was used to study the effect of interactions between Fe2+, Mg2+, and Mn2+ on the production of pullulanase. Box-Behnken showed a 1.1-fold increase (62.1 U/gds) in pullulanase production. The total increase in yield after all optimization was 6.37-fold. The present study reports for the first time the applicability of green gram husk as a potent substrate for pullulanase production by Penicillium viridicatum.

Waste management is a key issue for the agro-based industry. They are accountable for polluting the air and water resources as well as having severe impacts on human health because of their disposal practices. Various chemical treatments have traditionally been employed to handle solid waste. In recent times, more emphasis has been given to the biological conversion of these agro-wastes into useful products. Reports indicate that fungi can break down these complex organic compounds into simpler ones for their energy requirements 1 . Agro-wastes are a rich source of carbon that can be used to produce both microbial biomass and metabolites. It can act as a cheaper fermentation medium for lowering the cost of enzyme production 2 .
Advances in industrial biotechnology have the potential to make agro-industrial waste more economically valuable. Rice bran and wheat bran are important byproducts of the rice and wheat processing industries, respectively. These two byproducts can be effectively used to make a variety of high-value items 3 . Phaseolus vulgaris (local red kidney beans), Pistia stratiotes (water cabbage), Eichhornia crassipes (water hyacinth), and Ipomoea batatas (sweet potato) were identified as novel substrates to produce pullulanase 4 . Sugarcane bagasse, banana peel, rice bran, wheat bran, mausami peel, orange peel, legume husks, and other agro-industrial wastes have all been employed as substrates to produce biocatalysts 1,[5][6][7][8][9] . Green gram husk is rich in nutrients such as proteins Box-Behnken design at three levels for selected nutrients. The concentrations of the three independent factors that exhibited a significant effect (FeSO 4 , MnSO 4 , MgSO 4 ) on pullulanase production were opti- www.nature.com/scientificreports/ mized at three levels by the Box-Behnken design. The numerical optimization was carried out using the previously outlined approach 15 . The studied critical nutrients and their actual and coded values are given in Table 2.
Crude enzyme extraction and estimation of protein. The crude enzyme was extracted by flooding flasks with 1 mM phosphate buffer (pH 6.5) at room temperature (30 °C) for 15 min. after the fungus had grown to its maximal potential. The crude enzyme was separated from the substrate and biomass mixture by using a muslin cloth. To eliminate all the cells and debris, the extract was centrifuged at 4 °C for 15 min at 10,000 rpm in a cooling centrifuge 16 . The supernatant containing the crude enzyme was decanted and separated from the pellet and was used for the estimation of pullulanase activity.
Pullulanase activity estimation. Total pullulanase activity was measured by using the 3,5-dinitrosalicylic acid (DNS) method 17 . 0.5 mL of 1% (w/v) pullulan solution was mixed with 0.1 mL of enzyme sample and 0.4 mL of phosphate buffer (pH 6.5). The reaction mixture was kept at 40 °C for 30 min. Test tubes were incubated in a boiling water bath for 5 min. after adding 1 mL of DNS reagent. The liquid was then cooled to room temperature before adding 0.5 mL of a 1% (w/v) sodium potassium tartrate solution. The final volume was increased to 5 mL by adding 2.5 mL of double-distilled water. Using a UV-Vis Spectrophotometer, absorbance was measured at 570 nm (Shimazu-UV 1800, Japan). One unit of pullulanase was defined as the quantity of enzyme that released one micromole of glucose (reducing sugar equivalent) per minute at 40 °C and pH 6.5.

Results and discussion
The microorganism. The fungi used in the present study were isolated, screened, and identified in the previous study 9 . The sequence was submitted to GenBank under accession number MG672442. This isolate is most closely related to Penicillium viridicatum 9 .
Selection of agro-wastes for production of pullulanase in solid-state fermentation (SSF). One of the most important variables to consider is the selection of an appropriate agricultural residue as an SSF substrate. In SSF, different substrates have been screened for high yields of enzyme production 18 . When selecting a raw material in SSF, the availability and cost of the raw material are the two most important factors that need to be considered 19 . The chosen substrate facilitates the growth and development of microorganisms along with the synthesis of metabolites. In the present study, six major agricultural waste-based substrates such as wheat bran, green gram husk, red gram husk, black gram husk, banana peel, and mausambi peel were assessed for pullulanase production. Green gram husk had the highest enzyme activity (9.7 U/gds) of all the substrates studied ( Fig. 1), followed by red gram husk (7.29 U/gds) and wheat bran (5.52 U/gds). According to the findings of this study, pullulanase synthesis differed with a different kind of substrate due to differences in food supply and anchorage for growing cells. Proteins, lipids, carbohydrates, and minerals including iron, calcium, phosphorus, manganese, zinc, and copper are claimed to be abundant in the green gram husk 10 . There is no report on pullulanase production by Penicillium species in solid-state fermentation (SSF) using green gram husk as a substrate. Wheat bran was reported as a substrate for pullulanase production by Aspergillus flavus in SSF by Naik et al. 11 . The green gram husk was successfully used by Prakasham et al. 18 to produce the protease (9550 U/g biomass) by Bacillus species, but there are no reports on pullulanase production. This is the first time that green gram husk has been used as a substrate in SSF to produce pullulanase by Penicillium viridicatum.
Screening of important nutrients for green gram husk substrate using Plackett-Burman design. Plackett-Burman design (PBD) was previously used for quick screening of different nitrogen sources, growth/product promoters, minerals, and enzyme inducers for the synthesis of alpha-galactosidase by Aspergillus niger in a solid-state fermentation system 20 . Using shea butter cake as the major substrate, it was employed to efficiently identify essential medium components affecting Aspergillus niger lipase production 21 .
Similarly, PBD was used for screening nutrients for laccase production for Bacillus species 22 . Based on the above studies a total of 16 different nutrients and three dummy factors were used to screen the most effective nutrient to produce pullulanase by using the Plackett-Burman design. The number of experiments to be carried out based on the PBD design is n + 1, where n is the number of factors (variables). The high variables were designated as + 1 and the low variables as − 1 23,24 . The response of the 16 nutrients plus three dummy variables to the pullulanase production is given in  14 . Moreover, the green husk is rich in proteins 10 . The rest of the sixteen variables were not selected because they did not contribute www.nature.com/scientificreports/ significantly to pullulanase production at the selected level of confidence. A half-normal plot demonstrating the amplitude and orientations of standardized effects of major nutrients is shown in Fig. 2A. A half-normal probability plot is a graphical tool for determining which elements are important and which are not using these   www.nature.com/scientificreports/ ordered estimated effects. FeSO 4 is the furthest to the right of the response line, suggesting that it has the most positive impact on Penicillium viridicatum pullulanase production. Similarly, the order of relevance (MnSO 4 , FeSO 4 , and MgSO 4 ) of the variables influencing pullulanase production is depicted in the Pareto chart (Fig. 2B). Metal ions are essential in the production of enzymes 25 . Previously Reddy et al. 26 reported that FeSO 4 has a significant effect on pullulanase production by Clostridium thermosulfurogenes in SSF. Zhang et al. 27 showed that ferrous ions were required for enzyme synthesis, although their absence did not affect the growth of the culture. Similarly, MnSO 4 and MgSO 4 have also shown a positive effect on pullulanase yield. Manganese has been shown to significantly increase the synthesis of pullulanase and there was a 1.8-fold increase in pullulanase production by using MnSO 4 in production media 28,29 . Mn 2+ was found to increase enzyme production in earlier studies by other researchers 30,31 . A. flavus produced more enzymes when MgSO 4 .7H 2 O was added to the wheat bran medium 25 . Similarly, Kokab et al. 32 reported a higher yield of enzyme in SSF when the solid substrate was supplemented with MgSO 4 .

Box-Behnken design.
Based on the findings of the PBD, the Box-Behnken design was used to get the optimal concentration of chosen nutrients. Table 4 shows the nutrient levels as well as the outcomes. The results were entered into the program, and an ANOVA was run. Equation (1) depicts the regression model that resulted from the data analysis.
where Y is the yield, x 1 , x 2 , x 3 and are the concentrations of FeSO 4 , MnSO 4 , and MgSO 4 respectively. The R 2 value of 0.9922 demonstrated that the independent variables were responsible for 99.22% of the sample variations in pullulanase output, whereas just 0.78% of the total changes were not explained by the model. The Adjusted R 2 value of 0.9821 was likewise quite high, indicating that the model is very important. The CV value is 1.19% (a relatively low number), indicating that the studies were more accurate and efficient. The significant model terms are , and x 2 3 ( Table 5). The 3D response surface graphs and contour plots show how critical parameters interact and provide a visual representation of where the optimum conditions are located. Figure 3a-f was created for two parameters at a time, with the other variables maintained at their maximum value. The software's numerical optimization function was used to find the optimal levels (Design-Expert). During the optimization for the response, the variables FeSO4, MnSO4, and MgSO4 were placed in their ranges while the response (enzyme activity) was set to maximum level. The best option that met all of the aforementioned criteria and had overall desirability of 0.974 was found (FeSO 4 , 0.27%; MgSO 4 , 0.29%; MnSO 4 , 0.31%). Figure 3a,b depicts the interaction between MnSO 4 and FeSO 4 on the production of pullulanase. When the concentrations of MnSO 4 and FeSO 4 were increased, the curve of the graph showed a strong positive interaction with pullulanase production. The effect of MgSO 4 and FeSO 4 on pullulanase production was seen in Fig. 3c,d. As was seen in the www.nature.com/scientificreports/ graph, increasing the MgSO 4 concentration enhanced pullulanase production. In the case of FeSO 4 , a similar trend has been found. The interaction of MgSO 4 and MnSO 4 is shown in Fig. 3e,f, and optimal production may be attained at lower concentrations. This finding is in accordance with the cited literature 33,34 . In these publications, MgSO 4 at higher levels has been reported to reduce the production of enzymes. It has been reported previously that FeSO 4 , MgSO 4 , and MnSO 4 have a positive impact on enzyme production [35][36][37][38] . It may be due to activation, stability, simulation by these salts, and possible utilization of sulphate in protein synthesis 39,40 . In a study done by Alariya et al. 41 it was reported that manganese sulfate was the most suitable sulfate source. According to a study by Zhang et al. 42 Fe 2+ was found to be essential for enzyme production, although there was hardly any effect seen on cell growth in the absence of this ion.   www.nature.com/scientificreports/

Conclusion
It can be concluded that green gram husk is suitable as a substrate to produce pullulanase in SSF by Penicillium sp. When this substrate was further supplemented with FeSO 4 , MnSO 4 , and MgSO 4 , the total yield increased by 6.4 times. This is the first report on pullulanase production by Penicillium species using green husk as in substrate in SSF and its nutrient optimization.

Data availability
The datasets used and analyzed during the present study are available from the corresponding author on reasonable request.